Nitrile-Specific Protein NSP2 and Its Interacting Protein MPK3 Synergistically Regulate Plant Disease Resistance in Arabidopsis

Glucosinolates and their degradation products have a wide range of actions and are important components of plant defense. NSP2 (nitrile-specific protein 2) is a key regulator in the breakdown process of glucosinolates. However, the precise function of NSP2 in plant disease resistance beyond its role in glucosinolate degradation is still unclear. In this study, we discovered that NSP2 which was induced by Pst DC3000, influenced PR genes expression and reactive oxygen burst. Additionally, omics analysis revealed that NSP2 was engaged in plant-pathogen interaction and several hormone signal transduction pathways. Furthermore, immunoprecipitation-tandem mass spectrometry analysis (IP-MS), bimolecular fluorescence complementation (BiFC), and co-immunoprecipitation demonstrated that NSP2 interacts with MPK3. Genetic analysis shows that NSP2 may be a function downstream of MPK3. Upon pathogen inoculation, NSP2 protein levels increase while MPK3 protein levels decrease. Moreover, the level of phosphorylated NSP2 decreases. Taken together, this study sheds light on a new mode of synergistic action between NSP2 and MPK3 in the disease resistance process.


Introduction
Glucosinolates are nitrogen and sulfur-containing secondary metabolites found in Brassicales. Intact glucosinolates are thought to be physiologically inactive, whereas breakdown products play a critical role in plant defense against herbivores and pathogens [1][2][3][4][5][6]. Nitrile-specific proteins (NSPs) are a kind of particular protein involved in glucosinolate catabolism [7]. There are several mechanisms for the degradation of glucosinolates, and their breakdown into distinct products is influenced by a variety of circumstances, including the presence or lack of certain functional genes, such as ESP and NSP [8]. In Arabidopsis thaliana, there are five members of the NSP family, NSP1-NSP5, whose biological activities are poorly understood [7,9]. Except for NSP5, other members of the NSP family gene have 1-2 Jacalin structures in the N-terminus, the proteins containing this domain can respond to biological stresses like powdery mildew and gibberella infection, or abiotic stresses like cold injury, high salt, and drought, or hormones like SA, JA, and ABA [10,11]. This suggests that NSP family proteins may be involved in plant stress response. Furthermore, all NSP family proteins include four repeated kelch domains, which are hypothesized to be involved in protein interaction [12].
The mitogen-activated protein kinase (MAPK) cascade pathway is found in all species and is a highly conserved signaling mechanism. This route is essential for plant growth and development, as well as reaction to environmental stress. Activated MAPK serves three important physiological functions: (1) Phosphorylation of cytoskeletal proteins, resulting in cytoskeletal changes; (2) activation of other protein kinases to induce stress signal transmission; (3) translocation into the nucleus, binding to transcription factors, and activating them via phosphorylation, thereby regulating target gene expression. Numerous studies have highlighted a tight relationship between the MAPK signal and plant responses to abiotic stress, indicating that it plays an important role in enhancing plant stress resistance [13][14][15][16][17]. In the context of plant immunity, two MAPK cascades have been extensively studied. Both the MAP3K3/5-MKK4/5-MPK3/6 and MEKK1-MKK1/2-MPK4 cascades are rapidly and transiently activated upon perception of pathogen-associated molecular patterns (PAMPs), leading to gene reprogramming critical for mounting effective pathogen-associated molecular pattern-triggered immunity (PTI) responses [18][19][20][21].
The mechanisms of plant resistance and defense against pathogens mediated by MPK3 and MPK6 involve the regulation of transcriptional activation of defense genes, synthesis of plant antitoxins, cell-wall thickening, hypersensitivity, stomatal closure, endogenous hormone production, and reactive oxygen species (ROS) outbreak [22][23][24][25][26]. For example, HopAI1, a phosphothreonine lyase, can directly interact with and inactivate MPK3 and MPK6, thereby inhibiting gene expression, oxidative burst, and corpus callosum deposition induced by the activator flg22, resulting in increased susceptibility of plants to diseases [27]. In addition, MPK3 and MPK6 can regulate camalexin biosynthesis by phosphorylation of WRKY33 [28]. During the synthesis of camalexin, the indole glucosinolate production also increased, and participated in the immune process of Arabidopsis in response to pathogens [22]. Plant stomata play an essential role in innate immunity against bacterial invasion; it has been discovered that flg22-mediated stomatal closure is conveyed by Arabidopsis lipoxygenase 1 (LOX1) and MPK3/6 [24]. Active oxygen species (AOS) formed in response to stimuli and throughout development can operate as signaling molecules in eukaryotes, leading to particular downstream reactions. It was shown that MPK6 and MPK3 can be triggered by varying concentrations of H 2 O 2 in Arabidopsis. H 2 O 2 also activates an oxidation-signal-induced type 1 (OXI1) kinase, which is a key component of 1 O 2 and H 2 O 2 signaling and influences JA-dependent pathways and programmed cell death [29,30]. Exogenous H 2 O 2 treatment of Arabidopsis protoplasts can greatly boost the activities of MPK3 and MPK6; however, in the oxi1 mutant, the activity of MPK3/MPK6 was reduced when the protoplasts were treated with exogenous H 2 O 2 , and the resistance to downy mildew was decreased [29].
Due to the fact that simple nitrile formation upon tissue disruption depended almost entirely on NSP2 in seeds [31], it is necessary to reveal the function of NSP2 in the pathway of disease resistance. In this study, we revealed that NSP2 interacts with MPK3, and mutation or overexpression of NSP2 results in a susceptible phenotype when challenged with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), demonstrating the necessity for NSP2 to maintain a relatively stable expression level during disease resistance. Transcriptome and proteomic analysis revealed that NSP2 did respond to plant pathogen infection and plant hormone signal transduction. In addition, we predicted potential phosphorylation sites on NSP2 and confirmed its presence through observing phosphorylation modification of NSP2. Notably, after Pst DC3000 inoculation, the phosphorylation level of NSP2 was dramatically lowered, indicating a probable function for phosphorylation modification in NSP2 response to Pst DC3000. Furthermore, we found a substantial difference in MPK3 protein levels in NSP2 overexpression lines before and after inoculation with Pst DC3000, indicating that MPK3 may play a role in NSP2 phosphorylation. Our finding of an NSP2 interacting protein showed a new role for NSP2 in plant disease resistance. Overall, our results emphasize the significance of NSP2 and its post-translational changes in plant-pathogen interactions.

NSP2 Responds to Pst DC3000
Many studies suggest that NSP2 is involved in plant response to stress [8,31,32]. We explored whether Pst DC3000, a bacterial pathogen that causes strong plant defense responses, might regulate NSP2 expression. NSP2 expression indeed increased significantly in response to Pst DC3000 treatment, with the highest level of expression detected at 6 h post inoculation (Figure 1a), indicating that NSP2 is a component in the process of plant response to pathogen infection.
in response to Pst DC3000 treatment, with the highest level of expression detected at 6 h post inoculation (Figure 1a), indicating that NSP2 is a component in the process of plant response to pathogen infection.
Next, we obtained T-DNA insertion mutant nsp2 from Arabidopsis Biological Resources Center (ABRC) and NSP2 overexpression line NSP2-OE by Agrobacterium-mediated transformation. To test whether the NSP2 mutants have altered pathogen resistance, wild-type Col-0, nsp2, and NSP2-OE plants were inoculated with Pst DC3000. The total number of bacteria in nsp2 and NSP2-OE leaves were both significantly higher than that in Col-0 leaves at 2 days post inoculation (dpi), suggesting that both NSP2 lines exhibited inhibited resistance responses (Figure 1b).
Previous studies have shown that NSP2 is primarily expressed in seeds [31]. In order to further investigate the tissue-specific expression pattern of NSP2 in Arabidopsis, we analyzed the expression levels of NSP2 in different plant tissues. Consistent with previous reports, our results demonstrated that NSP2 was predominantly expressed in seeds, while it is also expressed in both roots and leaves (Figure 1c). Expression of NSP2 was detected by qRT-PCR at 0 h, 3 h, 6 h, and 12 h post Pst DC3000 treatment, respectively. Actin2 was used as an internal control. Transcript levels in Col-0 were set as 1. Three independent reactions were performed for each technical replicate. Two technical replicates were performed for each biological replicate. At least two biological replicates showed similar results. Error bars represent standard deviation and ** indicates p < 0.01 (paired student's t-test). (b) Measurement of bacterial titers of Pst DC3000. Col-0, nsp2 mutants, and NSP2-OE lines were infiltrated with a suspension of Pst DC3000 (OD600 = 0.002 in 10 mM MgCl2). Bacterial growth was determined at 0 h and 48 h. Each data point consisted of at least four samples. Experiments were repeated three times. Data represent the means of three replicates ± SDs (error bars). Different lowercase letters indicate a significant difference between different lines. Statistically significant differences were determined by one-way ANOVA and p < 0.05. (c) Tissue expression pattern of NSP2. Tissues of different periods were extracted from Col-0 for RNA extraction. Expression of NSP2 was detected by qRT-PCR, and Actin2 was used as an internal control. Three independent reactions were performed for each technical replicate. Two technical replicates were performed for each biological replicate. Error bars represent standard deviation. Different lowercase letters indicate a significant difference between different lines.

NSP2 Involved in the Plant Immune Signal Pathway at Transcriptional Level
To explore the disease resistance pathways involved in NSP2, we performed RNA sequencing (RNA-seq) analysis with wild type and NSP2-OE lines. Clean reads were Expression of NSP2 was detected by qRT-PCR at 0 h, 3 h, 6 h, and 12 h post Pst DC3000 treatment, respectively. Actin2 was used as an internal control. Transcript levels in Col-0 were set as 1. Three independent reactions were performed for each technical replicate. Two technical replicates were performed for each biological replicate. At least two biological replicates showed similar results. Error bars represent standard deviation and ** indicates p < 0.01 (paired student's t-test). (b) Measurement of bacterial titers of Pst DC3000. Col-0, nsp2 mutants, and NSP2-OE lines were infiltrated with a suspension of Pst DC3000 (OD 600 = 0.002 in 10 mM MgCl 2 ). Bacterial growth was determined at 0 h and 48 h. Each data point consisted of at least four samples. Experiments were repeated three times. Data represent the means of three replicates ± SDs (error bars). Different lowercase letters indicate a significant difference between different lines. Statistically significant differences were determined by one-way ANOVA and p < 0.05. (c) Tissue expression pattern of NSP2. Tissues of different periods were extracted from Col-0 for RNA extraction. Expression of NSP2 was detected by qRT-PCR, and Actin2 was used as an internal control. Three independent reactions were performed for each technical replicate. Two technical replicates were performed for each biological replicate. Error bars represent standard deviation. Different lowercase letters indicate a significant difference between different lines.
Next, we obtained T-DNA insertion mutant nsp2 from Arabidopsis Biological Resources Center (ABRC) and NSP2 overexpression line NSP2-OE by Agrobacterium-mediated transformation. To test whether the NSP2 mutants have altered pathogen resistance, wildtype Col-0, nsp2, and NSP2-OE plants were inoculated with Pst DC3000. The total number of bacteria in nsp2 and NSP2-OE leaves were both significantly higher than that in Col-0 leaves at 2 days post inoculation (dpi), suggesting that both NSP2 lines exhibited inhibited resistance responses (Figure 1b).
Previous studies have shown that NSP2 is primarily expressed in seeds [31]. In order to further investigate the tissue-specific expression pattern of NSP2 in Arabidopsis, we analyzed the expression levels of NSP2 in different plant tissues. Consistent with previous reports, our results demonstrated that NSP2 was predominantly expressed in seeds, while it is also expressed in both roots and leaves ( Figure 1c).

NSP2 Involved in the Plant Immune Signal Pathway at Transcriptional Level
To explore the disease resistance pathways involved in NSP2, we performed RNA sequencing (RNA-seq) analysis with wild type and NSP2-OE lines. Clean reads were mapped to Arabidopsis reference genome (TAIR10, www.arabidopsis.org); over 97% of the clean reads per library could be mapped to the reference database in the six individual  (Table S1). These results indicated that the RNA-seq data were sufficient for subsequent gene expression analysis.
Then, a total of 234 differentially expressed genes (DEGs) between WT (Col-0) and NSP2-OE were identified with >1.5-fold differences in expression and p-value < 0.05. Among these, 139 genes were upregulated and 95 genes were downregulated in NSP2-OE ( Figure 2a). To assess the correlation of gene expression patterns among the groups, systematic cluster analysis was performed and the heatmap result demonstrated the presence of numerous DEGs between WT and NSP2-OE (Figure 2b), indicating that the two materials marked disparities in gene expression patterns.
Among these, 139 genes were upregulated and 95 genes were downregulated in NSP2-OE ( Figure 2a). To assess the correlation of gene expression patterns among the groups, systematic cluster analysis was performed and the heatmap result demonstrated the presence of numerous DEGs between WT and NSP2-OE (Figure 2b), indicating that the two materials marked disparities in gene expression patterns.
The analysis of GO terms using the selected genes is useful in predicting the altered biological and molecular processes. Therefore, 234 DEGs were subjected to GO enrichment analysis to determine their functional significance in NSP2-OE. The significant enrichment was concentrated in several stress response biological processes, including iron ion starvation, cellular responses to hypoxia and decreased oxygen levels, as well as responses to chitin and aging ( Figure 2c). KEGG pathway analysis suggested the DEGs were mainly involved in spliceosome, plant-pathogen interaction, and plant hormone signal transduction ( Figure 2d). These results implied that NSP2 might play an important role in the response process of plants to pathogens. The analysis of GO terms using the selected genes is useful in predicting the altered biological and molecular processes. Therefore, 234 DEGs were subjected to GO enrichment analysis to determine their functional significance in NSP2-OE. The significant enrichment was concentrated in several stress response biological processes, including iron ion starvation, cellular responses to hypoxia and decreased oxygen levels, as well as responses to chitin and aging (Figure 2c). KEGG pathway analysis suggested the DEGs were mainly involved in spliceosome, plant-pathogen interaction, and plant hormone signal transduction (Figure 2d). These results implied that NSP2 might play an important role in the response process of plants to pathogens.
We tested the expression of four upregulated genes RAB18, STMP2, STMP7, SAUR13 and four downregulated genes MOP9, PDR7, SIKIC3, LRRAC1 to validate the RNAseq results. All four upregulated gene transcripts were significantly higher in NSP2-OE while the four downregulated genes showed lower expression in NSP2-OE than in Col-0 (Figure 3a,b). Among them, STMP2 and STMP7 are secretory transmembrane peptides unique to Brassicaceae, which function in plant growth and pathogen defense [33]. These results validated the RNA-seq data and confirmed that the NSP2 was related to plant-pathogen interaction. We tested the expression of four upregulated genes RAB18, STMP2, STMP7, SAUR13 and four downregulated genes MOP9, PDR7, SIKIC3, LRRAC1 to validate the RNA-seq results. All four upregulated gene transcripts were significantly higher in NSP2-OE while the four downregulated genes showed lower expression in NSP2-OE than in Col-0 ( Figure  3a,b). Among them, STMP2 and STMP7 are secretory transmembrane peptides unique to Brassicaceae, which function in plant growth and pathogen defense [33]. These results validated the RNA-seq data and confirmed that the NSP2 was related to plant-pathogen interaction. Actin2 was used as an internal control. Transcript levels in Col-0 were set as 1. Three independent reactions were performed for each technical replicate. Two technical replicates were performed for each biological replicate. Error bars represent standard deviation and * indicates p < 0.05; ** indicates p < 0.01 (paired student's t-test).

NSP2 Affect Several Pathogen-Related Protein Levels by Proteomic Analysis
We further elucidated the mechanism of NSP2 involvement in disease resistance response using proteomic analysis. A total of 439 and 521 differential proteins (Log2 (fold change) | ≥ 1, p < 0.05) were identified in NSP2-OE and nsp2 compared to Col-0, respectively. Among them, there were 206 upregulated and 233 downregulated proteins in NSP2-OE/Col-0, 247 upregulated and 274 downregulated proteins in nsp2/Col-0, respectively ( Figure 4a). Further analysis found that four proteins were upregulated in NSP2-OE and downregulated in nsp2 (AT1G10940, AT1G24280, AT2G29350, AT5G62440), while another four proteins were upregulated in nsp2 and downregulated in NSP2-OE (AT2G03870, AT3G01520, AT5G04420, AT5G08060). It is worth noting that two of these eight genes, AT1G10940 (SnrK2.4) and AT2G29350 (SAG13), have been clarified to participate in the response of plants to stress and diseases [34,35]. These eight genes should be closely linked to NSP2 and used as valuable targets for in-depth research.
GO enrichment analysis was carried out on the 439 and 521 differential proteins, respectively. The results showed that the differential proteins in NSP2-OE/Col-0 were mainly involved in the pathway of response to arsenic-containing substances, aging, and insect ( Figure 4b). However, the differential proteins in nsp2/Col-0 are mostly engaged in the response to desiccation, jasmonic acid biosynthetic process, and programmed cell death at biological process portion (Figure 4c). These findings implied that NSP2 plays a wide range of and complicated roles in plant regulation, potentially mainly impacting stress responses and resistant hormone signaling pathways. Col-0 and NSP2-OE lines. Actin2 was used as an internal control. Transcript levels in Col-0 were set as 1. Three independent reactions were performed for each technical replicate. Two technical replicates were performed for each biological replicate. Error bars represent standard deviation and * indicates p < 0.05; ** indicates p < 0.01 (paired student's t-test).

NSP2 Affect Several Pathogen-Related Protein Levels by Proteomic Analysis
We further elucidated the mechanism of NSP2 involvement in disease resistance response using proteomic analysis. A total of 439 and 521 differential proteins (Log2 (fold change) | ≥ 1, p < 0.05) were identified in NSP2-OE and nsp2 compared to Col-0, respectively. Among them, there were 206 upregulated and 233 downregulated proteins in NSP2-OE/Col-0, 247 upregulated and 274 downregulated proteins in nsp2/Col-0, respectively (Figure 4a). Further analysis found that four proteins were upregulated in NSP2-OE and downregulated in nsp2 (AT1G10940, AT1G24280, AT2G29350, AT5G62440), while another four proteins were upregulated in nsp2 and downregulated in NSP2-OE (AT2G03870, AT3G01520, AT5G04420, AT5G08060). It is worth noting that two of these eight genes, AT1G10940 (SnrK2.4) and AT2G29350 (SAG13), have been clarified to participate in the response of plants to stress and diseases [34,35]. These eight genes should be closely linked to NSP2 and used as valuable targets for in-depth research.

NSP2 Interacts with MPK3
In order to understand how NSP2 responds to Pst DC3000, NSP2 interacting proteins were detected by immunoprecipitation-tandem mass spectrometry analysis (IP-MS). MPK3 was co-precipitated with NSP2-GFP in NSP2 transgenic plants tagged with GFP and was chosen for further investigation since MAPK components are known to be involved in disease resistance.
The connection between NSP2 and MPK3 was established using a bimolecular fluorescence complementation (BiFC) assay. The vectors carried MPK3 fused with the YFP Cterminus and NSP2 fused with YFP N-terminus co-transform into N. benthamiana leaves. Bright fluorescence was observed in the nucleus and cytoplasm while no fluorescence was observed in the negative control. The findings validated NSP2's interaction with MPK3 ( Figure 5a). Co-immunoprecipitation (CoIP) assays obtained comparable results. Following the temporary co-expression of NSP2-MYC with MPK3-GFP in N. benthamiana, and the protein extract immunoprecipitation with anti-GFP antibodies, the band of NSP2-MYC was detected at the right position by Western blots (Figure 5b). GO enrichment analysis was carried out on the 439 and 521 differential proteins, respectively. The results showed that the differential proteins in NSP2-OE/Col-0 were mainly involved in the pathway of response to arsenic-containing substances, aging, and insect ( Figure 4b). However, the differential proteins in nsp2/Col-0 are mostly engaged in the response to desiccation, jasmonic acid biosynthetic process, and programmed cell death at biological process portion (Figure 4c). These findings implied that NSP2 plays a wide range of and complicated roles in plant regulation, potentially mainly impacting stress responses and resistant hormone signaling pathways.

NSP2 Interacts with MPK3
In order to understand how NSP2 responds to Pst DC3000, NSP2 interacting proteins were detected by immunoprecipitation-tandem mass spectrometry analysis (IP-MS). MPK3 was co-precipitated with NSP2-GFP in NSP2 transgenic plants tagged with GFP and was chosen for further investigation since MAPK components are known to be involved in disease resistance.
The connection between NSP2 and MPK3 was established using a bimolecular fluorescence complementation (BiFC) assay. The vectors carried MPK3 fused with the YFP C-terminus and NSP2 fused with YFP N-terminus co-transform into N. benthamiana leaves. Bright fluorescence was observed in the nucleus and cytoplasm while no fluorescence was observed in the negative control. The findings validated NSP2's interaction with MPK3 ( Figure 5a). Co-immunoprecipitation (CoIP) assays obtained comparable results. Following the temporary co-expression of NSP2-MYC with MPK3-GFP in N. benthamiana, and the protein extract immunoprecipitation with anti-GFP antibodies, the band of NSP2-MYC was detected at the right position by Western blots (Figure 5b).

NSP2 and MPK3 Worked Synergistically in the Process of Disease Resis
To study the role of NSP2 and MPK3 in regulating plant immunity double mutant nsp2mpk3 by crossing T-DNA insertion mutants of NSP notypic analysis revealed that the nsp2mpk3 double mutant displayed s to the nsp2 single mutant, both slightly smaller than the wild type (Fig  that NSP2 may be a function downstream of MPK3. The bacterial grow inoculation of nsp2mpk3 double mutant was higher than that in Col-0, to mpk3 single mutant (Figure 6b). After Pst DC3000 inoculation, the disease-resistant related genes showed that PR1, PR2, and PR5 were i degrees in each line and the expression levels of each gene in nsp2, m NSP2-OE were lower than that in Col-0 (Figure 6c-e). These results in and MPK3 share a pathway and synergistically promote disease resista

NSP2 and MPK3 Worked Synergistically in the Process of Disease Resistance
To study the role of NSP2 and MPK3 in regulating plant immunity, we generated the double mutant nsp2mpk3 by crossing T-DNA insertion mutants of NSP2 and MPK3. Phenotypic analysis revealed that the nsp2mpk3 double mutant displayed similar phenotypes to the nsp2 single mutant, both slightly smaller than the wild type (Figure 6a), suggesting that NSP2 may be a function downstream of MPK3. The bacterial growth with Pst DC3000 inoculation of nsp2mpk3 double mutant was higher than that in Col-0, which was similar to mpk3 single mutant (Figure 6b). After Pst DC3000 inoculation, the expression level of disease-resistant related genes showed that PR1, PR2, and PR5 were induced to varying degrees in each line and the expression levels of each gene in nsp2, mpk3, nsp2mpk3, and NSP2-OE were lower than that in Col-0 (Figure 6c-e). These results indicated that NSP2 and MPK3 share a pathway and synergistically promote disease resistance in plant.
to mpk3 single mutant (Figure 6b). After Pst DC3000 inoculation, the expression level disease-resistant related genes showed that PR1, PR2, and PR5 were induced to varyi degrees in each line and the expression levels of each gene in nsp2, mpk3, nsp2mpk3, a NSP2-OE were lower than that in Col-0 (Figure 6c-e). These results indicated that NS and MPK3 share a pathway and synergistically promote disease resistance in plant. and Pst DC3000 treatment, respectively. Actin2 was used as an internal control. Transcript levels in Col-0 treated with H 2 O were set as 1. Three independent reactions were performed for each technical replicate. Two technical replicates were performed for each biological replicate. Error bars represent standard deviation. Different lowercase letters indicate significant differences between different lines. Statistically significant differences were determined by one-way ANOVA and p-values were < 0.05.
Since reactive oxygen species (ROS) are key signal molecules that allow cells to respond quickly to varied stimuli and play an important role in plant defense responses, we performed DAB and NBT staining on Col-0, nsp2, NSP2-OE, mpk3, and nsp2mpk3 leaves after inoculation with Pst DC3000. The staining area of DAB and NBT in nsp2, NSP2-OE, mpk3, and nsp2mpk3 leaves after invasion of Pst DC3000 were significantly reduced compared with Col-0 (Figure 7a-c), indicating that the outbreak of ROS after invasion of Pst DC3000 was reduced compared with that of Col-0, which was consistent with the susceptible phenotype observed following Pst DC3000 inoculation.
Aniline blue staining was also used to detect callose deposition in leaves of Col-0, nsp2, NSP2-OE, mpk3, and nsp2mpk3 after Pst DC3000 inoculation (Figure 7d). A large number of fluorescence signals could be detected in wild type; however, the fluorescence signals of nsp2, NSP2-OE, mpk3, and nsp2mpk3 were very few (Figure 7e), indicating that the callose content of nsp2, NSP2-OE, mpk3, and nsp2mpk3 was lower than that of Col-0, which was consistent with the fact that nsp2, NSP2-OE, mpk3, and nsp2mpk3 were sensitive to Pst DC3000.
Aniline blue staining was also used to detect callose deposition in leaves of Col-0, nsp2, NSP2-OE, mpk3, and nsp2mpk3 after Pst DC3000 inoculation (Figure 7d). A large number of fluorescence signals could be detected in wild type; however, the fluorescence signals of nsp2, NSP2-OE, mpk3, and nsp2mpk3 were very few (Figure 7e), indicating that the callose content of nsp2, NSP2-OE, mpk3, and nsp2mpk3 was lower than that of Col-0, which was consistent with the fact that nsp2, NSP2-OE, mpk3, and nsp2mpk3 were sensitive to Pst DC3000. The leaves of Col-0, nsp2, mpk3, mpk3nsp2, and NSP2-OE inoculated with Pst DC3000 were cut and placed in the NBT or DAB staining solution, treated in the dark at room temperature overnight, and photographed after decolorization. (b,c) Quantification of the NBT and DAB staining area in the leaves was statistically analyzed by the ImageJ software. Error bars represent standard deviation. Statistically significant differences were determined by one-way ANOVA and ** indicates p-values were < 0.01. (d) The leaves of Col-0, nsp2, mpk3, mpk3nsp2, and NSP2-OE inoculated with Pst DC3000 were cut and soaked in 95% alcohol. Until the leaves fade and become transparent, they were transferred to the dye solution (150 mmol/L K 2 HPO 4 , pH = 9.5, 0.01% aniline blue) for 30 min, washed with sterile water, and then observed under a slide fluorescence microscope. (e) Quantification of callose in the visual field area was statistically analyzed by the ImageJ software. Error bars represent standard deviation. Statistically significant differences were determined by one-way ANOVA and ** indicates p-values were < 0.01.

Pathogenic Inoculation Alters the NSP2 Phosphorylation Status
We discovered that Pst DC3000 induces NSP2 transcriptional expression (Figure 1a), and that NSP2 protein content is likewise enhanced after pathogen treatment (Figure 8a). We used anti-MPK3 antibodies in above ground portions of nsp2 mutant and NSP2-OE before and after Pst DC3000 treatment to examine the influence of NSP2 on MPK3 abundance. Interestingly, we detected a large rise in MPK3 abundance in NSP2-OE at normal condition; however, following Pst DC3000 treatment, the increasing trend disappeared and became similar to the wild type (Figure 8b). MPK3 is a protein kinase that typically conveys signals via phosphorylation during signal transduction. As a result, we postulated that MPK3 could phosphorylate NSP2. Based on the amino acid sequences of NSP2, we predicted that several high confidence sites in NSP2 could probably be phosphorylated by MPK3 (Table S2). Following that, we used phos-tag gel assay to look at the phosphorylation alteration of NSP2. Surprisingly, we observed that NSP2 had phosphorylation modification; however, it was drastically reduced following Pst DC3000 treatment (Figure 8c), indicating that pathogen challenge may have modified the phosphorylation state of NSP2. Our data suggest that pathogenic inoculation alters the NSP2 phosphorylation status which might be caused by MPK3.
we predicted that several high confidence sites in NSP2 could probably be phosphoryl by MPK3 (Table S2). Following that, we used phos-tag gel assay to look at the phosp ylation alteration of NSP2. Surprisingly, we observed that NSP2 had phosphoryla modification; however, it was drastically reduced following Pst DC3000 treatment (Fi 8c), indicating that pathogen challenge may have modified the phosphorylation sta NSP2. Our data suggest that pathogenic inoculation alters the NSP2 phosphorylation tus which might be caused by MPK3.

Discussion
Pst DC3000 is one of the most common and dangerous plant pathogens. It may infect not only Arabidopsis thaliana, but also many commercial crops, such as tomato and N. benthamiana, resulting in plant leaf damage and massive economic losses. The interaction between Pst DC3000 with the model plant Arabidopsis thaliana has become a valuable tool for studying the molecular mechanisms of plant disease resistance [36]. We discovered that Pst DC3000 induced NSP2 expression, and that lack or overexpression of NSP2 in plants resulted in higher sensitivity to Pst DC3000 (Figure 1a,b and Figure 6b), emphasizing the necessity of maintaining constant levels of NSP2 during pathogen infection. The importance of protein homeostasis in disease resistance has been previously reported. Both overexpression and suppression of OsLSR lead to the autoactivation of immune response in rice [37], indicating that the transcript level of some key genes is strictly regulated by the immune system in plants.
Through transcriptome and proteomic analysis, we discovered that NSP2 participates in mRNA splicing, senescence, pathogen interaction, and hormone signal transduction (Figure 4), indicating that NSP2 plays an important role in the control of plant growth, development, and immunological responses. Functional studies of NSP family genes in Arabidopsis are currently uncommon. According to bioinformatic prediction, stress-related cis-regulatory elements are commonly found in the promoter region of NSP family genes, and NSP5 can considerably upregulate its expression under drought stress [38]. The structural variations in NSP proteins, as well as their enzyme activity in catalyzing the breakdown of glucosinolates, show that NSP family genes operate differently [8]. As a result, more studies on disease resistance of other genes in the NSP family are required.
The MAPK cascade pathway has been extensively investigated, demonstrating its critical function in plant resistance and defense response [39][40][41]. The MAPK cascade route associated with plant disease resistance has a large number of members at various levels, and these members may have cross-effects with other MAPK cascade pathways. In this work, we highlight the significance of NSP2 in controlling plant disease resistance by identifying its interacting proteins, and we discovered that NSP2 connected with MPK3. The nsp2mpk3 double mutant showed a developmental behavior comparable to nsp2 (Figure 6a), suggesting that NSP2 is genetically downstream of MPK3. Following Pst DC3000 inoculation, the double mutant demonstrated sensitive symptoms (Figure 6b). These findings indicate that we discovered a novel component for the MPK3 function complex in the immune signal transduction.
MPK3's main role is to influence the expression of downstream genes and the regulation of physiological metabolism by phosphorylating downstream target proteins, which in turn affects plant growth and development and stress adaption [15,42,43]. We predicted NSP2 phosphorylation sites and postulated that NSP2 may be phosphorylated by MPK3, affecting its biological activity. Phosphorylation of NSP2 was indeed detected using phostag gel, and the modification was dramatically decreased following Pst DC3000 treatment (Figure 8b). Although no direct evidence relating NSP2 phosphorylation to MPK3 was revealed, our study laid the groundwork for future research into the potential of NSP2 being a direct target of MPK3. Therefore, we propose this possible regulatory pathway: under normal conditions, MPK3 phosphorylates NSP2 and phosphorylated NSP2 function to resist the infection of pathogens; upon treatment with Pst DC3000, the protein level of MPK3 decreases in NSP2-OE, resulting in a decrease in the phosphorylation level of NSP2, thus showing a susceptible phenotype; however, the cause of the reduction in MPK3 protein is uncertain. Of course, there is another possibility: after challenge by the pathogen, another unknown dephosphorylase was activated to interact with NSP2, resulting in a decrease in NSP2 protein phosphorylation.
Overall, our study identifies a new role for NSP2 in pathogen resistance, sheds light on the function of NSP2 in plant-pathogen interactions, and establishes a connection between NSP2 and MPK3. Our result advances the understanding of plant defense mechanisms and provides a foundation for future research on its role in disease resistance modulation in Arabidopsis.

Plant Material and Growth Conditions
Genotypes used in this study were Col-0, nsp2 (SALK_057194C), and mpk3 (SALK_100651), the mutants were ordered from Arabidopsis Biological Resources Center (ABRC). Double mutant was created by genetic crosses, and F2 plants were genotyped for homozygotes. Transgenic plants were generated by the floral dip method. Surface-sterilized seeds were plated onto half-strength Murashige and Skoog medium (1/2 MS) for 7 days and then moved to soil and cultured in a growth chamber for 4-5 weeks (23 • C, 16/8 h light/dark). The illumination intensity was 90 µmol/m 2 /s.

Bacterial Cultivation and Inoculation
Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) was grown as previously described [44]. Bacterial cells were rinsed with water, diluted in 10 mM MgCl 2 (OD 600 = 0.002), then infiltrated into Arabidopsis leaves. Thereafter, 4-week-old Arabidopsis plants grown in soil were sprayed with Pst DC3000 (OD 600 = 0.2) or H 2 O (mock) for 24 h to examine resistance-related gene expression and reactive oxygen species accumulation. The samples were collected individually and kept at −80 • C until needed.

Reactive Oxygen Species Accumulation Determination
Reactive oxygen species (ROS) accumulation was measured by DAB (3,3 -diaminobenzidine) and NBT staining as previously described [45]. The leaves were darkened and kept at room temperature overnight after being placed in the staining solution (NBT dissolved in 25 mM KH 2 PO 4 (pH = 7.6); the final concentration was 0.1 g/L). The staining solution was discarded, and the leaves were decolorized by boiling in the fixing solution (lactic acid: glycerol: ethanol = 1:1:3). The leaves were then placed in the new fixing solution, and images were taken after they had cooled.

Plasmid Construction
The full-length NSP2 cDNA was amplified by RT-PCR and ligated to pENTR/Dtopo vector (Thermo Fisher Scientific, K240020, Waltham, MA, USA). The pENTR-NSP2 plasmid was transferred into the binary vector pMDC83 to produce p35S::NSP2-GFP by LR recombination (Thermo Fisher Scientific, 11791-100). To generate the cCFP-MPK3 and nVenus-NSP2 fusion vector, the MPK3 and NSP2 cDNA were first cloned into the pSAT4-cCFP-C and pSAT1-nVenus-C vector, respectively. The DNA fragment containing cCFP-MPK3 and nVenus-NSP2 was released by I-SceI and AscI restriction enzyme, respectively and subsequently cloned into the pPZP-RCS2-ocs-bar vector [46]. All the primers are listed in Table S3.

Bimolecular Fluorescence Complementation Assay
BiFC assays were performed as described by Lee et al. [46]. Paired cCFP and nVenus constructs were co-infiltrated in N. benthamiana leaves for 48 h. YFP signals were then detected with a confocal microscopy LSM 880 CLSM (Zeiss, Jena, Germany) at 488 nm.

Quantitative Real-Time PCR
The total RNA was extracted from the frozen Arabidopsis leaf samples with RNAiso Plus (Takara 9101). The resulting RNA samples were treated with DNase I (TransGen Biotech GD201-01) followed by reverse transcription using HiScript II Reverse Transcriptase (Vazyme R223-01) according to the manufacturer's instructions. qRT-PCR was performed in triplicate using SYBR Green Real-Time PCR Master Mix (Vazyme Q711-02) on the Bio-Rad CFX96 system. The 2 − ∆∆ Ct method was used to calculate the relative gene expression level across the samples. Finally, the results were presented as histograms by GraphPad Prism 8 software (GraphPad, San Diego, CA, USA). Primers used are listed in Table S3.

RNA Sequencing
Total RNA was extracted from samples of different genotypes. Sequencing libraries were constructed by Novogene (Beijing, China), with PE150 mode sequencing being performed on the Illumina sequencer. Prior to sequence alignment, the obtained reads were